The goal of this proposal is to gain insight into the specification and regulation of eukaryotic origins of DNA replication. Our model organism, Tetrahymena thermophila, harbors a 21 kb ribosomal DNA (rDNA) minichromosome that is amplified 9,000-fold during development, but is replicated once per cell cycle during vegetative cell divisions. We have identified trans-acting factors that repress or activate rDNA replication by binding to a common cis-acting replication determinant, the type I element. We recently discovered that the Tetrahymena Origin Recognition Complex (ORC) contains an integral RNA subunit that forms Watson:Crick Dase pairs with the type I element T-rich strand. Remarkably this RNA, designated 26T, corresponds to the extreme 3' terminus of 26S ribosomal RNA. We hypothesize that 26T RNA is processed from cytoplasmic ribosomes and functions as a physiological link between DNA replication and cellular metabolism. We further speculate that RNA-mediated recognition is restricted to the rDNA replicon and provides a mechanism for selectively targeting ORC to the rDNA origin during programmed gene amplification. In this application, we propose experiments to determine the protein composition of this remarkable ORC complex. We will examine the contribution 26T RNA residues to rDNA sequence recognition and origin activation, and will determine how ORC recognizes other replication origins that lack binding sites for 26T.RNA. We will test models for 26T RNA biogenesis to gain insights into regulation, and will introduce chimeric ARS1/rDNA replicons into the developing macronucleus to examine the molecular basis for programmed gene amplification. For these studies, we will employ powerful biochemical and reverse genetic approaches, with the goal of elucidating underlying mechanisms that regulate the initiation of eukaryotic DNA replication. The proposed experiments for studying this new replication paradigm, RNA-mediated origin recognition, should provide exciting new insights into chromosome biology. The unprecedented discovery of a role for ribosomal RNA in DNA replication indicates that these studies will have broad implications in and beyond the replication field. [unreadable] [unreadable] [unreadable]